Neurons in the brain have limited energy reserves and thus rely on a just-in-time delivery strategy in which active neurons signal to the brain microvasculature to locally dilate and increase regional cerebral blood flow (CBF), resupplying nutrients and oxygen as well as removing toxic metabolites. Despite extensive study, the mechanisms underlying the functional linkage between neuronal demand and vascular supply, termed neurovascular coupling (NVC), remain poorly understood. CBF within the brain is mediated by parenchymal arterioles (PAs) and hundreds of miles of capillaries, which enormously extend the territory of perfusion. Capillaries are the proximate vascular elements for the vast majority of neurons; thus, the dense capillary network can be viewed as a sensory web, detecting the rhythm of neural activity. Notably, neuronal activity- dependent increases in CBF at the surface of the brain are conducted at a rate that can only be explained by an electrical signal. We propose that strong inward-rectifier K+ (Kir2) channels in the vascular endothelium of capillaries, as well as arterioles, serve as exquisite sensors of local external K ([K ]e)-a byproduct of neuronal activity-that transmit an electrical signal to cause upstream dilation of PAs and surface arteries, and coordinate blood flow within the brain. Our extensive preliminary data, including the discovery that capillary endothelial cells (ECs) express robust Kir currents, as do both smooth muscle cells (SMC) and ECs of PAs, support this. We further propose that Kir-dependent electrical signals are augmented/sustained by slower retrograde Ca signals mediated by EC TRPV4 channels and G protein-coupled receptors of the Gq/11 type. We deploy a wide variety of novel, state-of-the-art experimental approaches using intact animals, native tissue and freshly isolated cells, complemented by sophisticated computational modeling, to test these ideas. Aim 1 will explore the impact of dual expression of Kir channels in EC and SMC compartments on K+-sensing, vascular dynamics, and the efficacy of EC-dependent vasodilators. Aim 2 will provide the first insights into ion channel properties and electrical and Ca2+-signaling in brain capillary endothelium using our newly developed pressurized arteriole-capillary ex vivo preparation. Building on Aims 1 and 2, Aim 3 will use in vivo approaches to explore inside-out electrical and Ca2+ signaling from capillaries to upstream arterioles and surface arteries in the context of NVC. The proposed work has the potential to revolutionize our understanding of communication within the brain microcirculation, and as such will provide the foundation for understanding small vessel diseases of the brain.